Cells are organized in a highly complex manner. And while there are many different types of cells - each organized in a different manner according to their function - they do share certain commonalities. Among these commonalities are membranes that functions not only as a barrier between the extra- and intra-cellular environment, but is also involved in many cell-biological processes like signaling and transport; and since each of these processes require a different set of components and organization, a large heterogeneity is expected within these membranes. Electron microscopic techniques have been invaluable to our understanding of the complex cellular architecture (and the corresponding dynamics). In recent years, 3D electron microscopic techniques like electron tomography and the focussed ion-beam scanning electron microscope have emerged and provided us with an exciting new perspective of cellular structures and their inter- and intra-organellar relationships. However, extracting relevant information from (3D) micrographs heavily relies on manual segmentation techniques and is therefore affected by the eye of the beholder. In the work presented in this thesis we have explored template matching as an approach to harvest biologically relevant information from tomographic volumes in a (semi-)objective manner. By using a stylized cuboid-shaped template we could extract membranes from the tomographic volumes and thus study the overall morphology of organelles. But more importantly, by comparing the matching results of slightly distinct templates, we were able to visualize ultra-structural details that are lost by manual segmentation. By applying this approach we up the ante of electron tomographic approaches and were able to bring it into a new realm where we can study and visualize in three dimensions the membrane-organization of biologically functional and significant events like lipid- and protein-domains
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